The subject of the present invention is a new cysteine-rich peptide called heliomicine, its use as a medicarnent and the compositions containing it, a DNA sequence encoding this peptide, a vector containing it for the transformation of a host organism and the method of transforming the said organism.
The invention relates more particularly to the transformation of plant cells and of plants, the heliomicine produced by the transformed plants conferring on them resistance to diseases, in particular of fungal origin.
There is currently an increasing need to make plants resistant to diseases, in particular fungal diseases, in order to reduce or even avoid having to use treatments with antifungal protection products, in order to protect the environment. One means of increasing this resistance to diseases consists in transforming plants so that they produce substances capable of providing their defense against these diseases.
In the field of human health, opportunistic fungal infections exist for which no truly effective treatment currently exists. In particular, this is the case for certain serious invasive mycoses which affect hospital patients whose immune system is suppressed following a transplant, a chemotherapy or human immunodeficiency virus (HIV) infection. Compared with the antimicrobial agent arsenal, the current range of antifungal agents is very limited. A real need therefore exists to characterize and develop new classes of antifungal substances.
Various substances of natural origin, in particular peptides, are known which exhibit bactericidal or fungicidal properties, in particular against the fungi responsible for plant diseases. However, a first problem consists in finding such substances which not only can be produced by transformed plants, but which can still preserve their bactericidal or fungicidal properties and confer them on the said plants. For the purposes of the present invention, bactericidal or fungicidal is understood to mean both the actual bactericidal or fungicidal properties and the bacteriostatic or fungistatic properties.
Cysteine-rich peptides are also known which exhibit bactericidal or bacteriostatic activities, but which do not exhibit fungicidal or fungistatic activity. Another problem consists in finding a cysteine-rich peptide which exhibits a high fungicidal or fungistatic activity compared with the peptides of the state of the art.
Heliomicine is a peptide isolated from the haemolymph of the lepidopteron Heliothis virescens which exhibits fungicidal activity against the fungi responsible for plant diseases and the fungi of human or animal pathology. After having first synthesized the gene for heliomicine, it was also found that it could be inserted into a host organism, such as a yeast or a plant, so as to express heliomicine and either produce purified or nonpurified heliomicine, or confer on the said host organism properties of resistance to fungal diseases, providing a particularly advantageous solution to the problems set out above.
The subject of the invention is therefore first heliomicine, its use as a medicament or in agrochemistry for the protection of plants, the compositions comprising it, a nucleic acid fragment encoding heliomicine, a chimeric gene comprising the said fragment encoding heliomicine as well as heterologous regulatory elements at the 5′ and 3′ positions which can function in a host organism, in particular in yeasts or plants and a vector for transforming the host organisms containing this chimeric gene, and the transformed host organism. It also relates to a transformed plant cell containing at least one nucleic acid fragment encoding heliomicine and a plant resistant to diseases containing the said cell, in particular which is regenerated from this cell. It finally relates to a method of transforming plants to make them resistant to diseases into which a gene encoding heliomicine is inserted by means of an appropriate vector. It finally relates to a method of preparing heliomicine by transformed host organisms. p Heliomicine is understood to mean according to the invention any peptide comprising essentially the peptide sequence of formula (I) below,Xaa-Cys-Xab-Cys-Xac-Cys-Xad-Cys-Xae-Cys-Xaf-Cys-Xag  (I)in which:    Xaa is —NH2 or a peptide residue comprising from 1 to 10 amino acids, preferably from 1 to 6 amino acids,    Xab is a peptide residue comprising from 1 to 1510 amino acids, preferably 10,    Xac is a peptide residue of 3 amino acids,    Xad is a peptide residue comprising from 1 to 9 amino acids, preferably 9,    Xae is a peptide residue comprising from 1 to 7 amino acids, preferably 7,    Xaf is a peptide residue of 1 amino acid, and    Xag is —OH or a peptide residue comprising from 1 to 5 amino acids, preferably 1 or 2 amino acids.
According to a preferred embodiment of the invention, Xaa comprises at least one basic amino acid, and/or Xad comprises at least one basic amino acid. Advantageously, Xad comprises I, 2, 3 or 4 basic amino acids.
Advantageously, Xad represents the following peptide sequence -Lys-Xad′-Xad″-Gly-His-, in which Xad represents a peptide residue of 1 basic amino acid and Xad″ represents a peptide residue comprising from a to 5 amino acids, preferably 5.
Basic amino acids are understood to mean more particularly according to the invention the amino acids chosen from lysine, arginine or homoarginine.
Preferably, Xad represents the folowing peptide sequence -Lys-Arg-Arg-Gly-Tyr-Lys-Gly-Gly-His- (SEQ ID NO:41) or Leu-Leu-Arg-Gly-Tyr-Lys-Gly-Gly-His- (SEQ ID NO49).
According to another preferred embodiment of the invention, Xac comprises at least one acidic amino acid, preferably one.
Advantageously, Xac represents the following peptide sequence -Asn-Xac′-Xac″-, in which Xac represents a peptide residue of 1 amino acid, and Xac represents a peptide residue of 1 acidic amino acid.
Acidic amino acid is understood to mean according to the invention any amino acid comprising on a side chain an organic acid function, more particularly a carboxylic acid preferably chosen from glutamic acid (Glu) or aspartic acid (Asp).
Preferably, Xac represents the following peptide sequence -Asn-Gly-Glu- (SEQ ID NO:50) or Ala-Ala-Glu- (SEQ ID NO:51).
Advantageously, Xaa represents the following peptide sequence Xaa′-Gly-Xaa″- in which Xaa′ represents NH2 or a peptide residue comprising 1 to 9 amino acids, preferably 1 to 5 amino acids, and Xaa″ represents a peptide residue comprising at least one amino acid, preferably chosen from Leu, Ile, Val, Pro, Ser or Thr, and/or Xab represents the following peptide sequence -Val-Xab′-Asp-, in which Xab′ represents a peptide residue comprising from 0 to 8 amino acids, preferably 8, and/or
Xae represents the following peptide sequence-Gly-Xae′-Asn-, in which Xae′ represents a peptide residue comprising from 0 to 5 amino acids, preferably, and/or
Xaf represents one of the following amino acids -Trp-,Phe, Leu, Ile or Val and/or
Xag represents the following peptide sequence -Glu-Xag′ in which Xag′ represents OH or a variable residue having a sequence comprising from 1 to 4 amino acids, preferably 1 amino acid.
According to a more preferred embodiment of the invention, Xaa represents the following peptide sequence NH2-Asp-Lys-Leu-Ile-Gly-Ser- (SEQ ID NO:46) or NH2-Ala-Ala-Ala-Ala-Gly-Ser-, (seq ID NO:52) and/or Xab represents the following peptide sequence -Val-Trp-Gly-Ala-Val-Asn-Tyr-Thr-Ser-Asp- (SEQ ID NO:47), and/or Xae represents the following peptide sequence -Gly-Ser-Phe-Ala-Asn-Val-Asn- (SEQ ID NO:48), and/or Xaf represents the following amino acid -Trp- and/or Xag represents the following peptide sequence -Glu-Thr-OH or -Arg-Thr-OH.
According to a more preferred embodiment of the invention, the heliomicine is the peptidc represented with its coding sequence by the sequence identifier No. 2 (SEQ ID NO:2). The same sequence is described, corresponding to amino acids 6 to 49 of the sequence identifier No. 1 (SEQ ID NO:1) with a different coding sequence.
The NH2-terminal residue may exhibit a post-translational modification, for example an acetylation, likewise the C-terminal residue may exhibit a post-translational modification, for example an amidation.
Peptide sequence comprising essentially the peptide sequence of general formula (I) is understood to mean not only the sequences defined above, but also such sequences comprising at either of their ends, or at both ends, peptide residues necessary for their expression and targeting in a host organism. Host organism is understood to mean any organism comprising at least one cell, whether microorganisms, in particular a yeast or a bacterium, or alternatively plant cells or alternatively higher organisms such as plants.
This may be in particular a “peptide-heliomicine” fusion peptide whose cleavage by the enzymatic systems of the host organism allows there lease of heliomicine, heliomicine being defined above. The peptide fused with heliomicine may be a signal peptide or a transit peptide which makes it possible to control and orient the production of heliomicine in a specific manner in a portion of the host organism, such as for example the cytoplasm, the cell membrane, or in the case of plants in a particular type of cell compartment or of tissues or in the extracellular matrix.
According to one embodiment, the transit peptide may be a signal for chloroplast or mitochondrial homing, which is then cleaved in the chloroplasts or the mitochondria.
According to another embodiment of the invention, the signal peptide may be an N-terminal signal or “prepeptide”, optionally in combination with a signal responsible for retaining the protein in the endoplasmic reticulum, or a peptide for vacuolar homing or “propeptide”. The endoplasmic reticulum is the site where the operations for processing the protein produced, such as for example the cleavage of the signal peptide, are performed by the “cellular machinery”.
The transit peptides may be either single, or double, and in this case optionally separated by an intermediate sequence, that is to say comprising, in the direction of transcription, a sequence encoding a transit peptide of a plant gene encoding a plastid localization enzyme, a portion of sequence of the N-terminal mature part of a plant gene encoding a plastid localization enzyme, and then a sequence encoding a second transit peptide of a plant gene encoding a plastid localization enzyme, as described in application EP 0 508 909.
As transit peptide useful according to the invention, there may be mentioned in particular the signal peptide of the tobacco pahtogen-related protein 1α (PR-1α) gene described by Cornelissen et al., represented with its coding sequence by the sequence identifier No. 2 (SEQ ID NO:2), in particular when heliomicine is produced by plant cells or plants, or the precursor of factor Mat α1 when heliomicine is produced in yeasts.
The fusion peptide “MFα1/heliomicine” with the five residues of the propeptide of factor MFα1(Ser-Leu-Asp-Lys-Arg) (SEQ ID NO:53), which are situated at the N-terminal position, and its coding sequence are part the present invention, described in particular by the sequence identifier No. 1 (SEQ ID NO:1), corresponding to amino acids 1 to 49.
The “PR-1α signal peptide-heliomicine” fusion peptide and its coding sequence are also part of the present invention, described in particular by the sequence identifier No. 3 (SEQ ID NO:3).
The fusion peptide comprising the signal peptide of the maize polygalacturonase PG1. gene fused with heliomicine “PG1 signal peptide/heliomicine” is represented with its coding sequence by the sequence identifiers Nos. 18 and 20 (SEQ ID NO:18 and SEQ ID NO:20).
According to a preferred embodiment of the invention, the cysteine residues of the peptide of formula (I) form at least one intramolecular disulphide bridge, preferably three disulphide bridges. According to a preferred embodiment of the invention, the disulphide bridges are established between the cysteine residues 1 and 4, 2 and 5, and 3 and 6.
Heliomicine is a peptide which is particularly active against fungi and yeasts, and may as such be used preventatively or curatively to protect various organisms against fungal attacks. The present invention therefore relates to heliomicine as a medicament. It also relates to the use of heliomicine for the treatment of plants against fungal attacks, by applying heliomicine directly to the said plants.
The present invention also relates to a composition comprising heliomicine and an appropriate vehicle. The first quality of the appropriate vehicle is not to substantially degrade the heliomicine in the composition, and not to reduce the bactericidal and fungicidal properties of the heliomicine. This composition may be a cosmetic composition and in this case the appropriate vehicle is cosmetically acceptable(suitable in addition for application to the skin or the exoskeleton), or a pharmaceutical composition for a therapeutic use and in this case the appropriate vehicle is pharmaceutically acceptable, appropriate for administration of heliomicine by the topical route per os or by injection, or alternatively an agrochemical composition and in this case the appropriate vehicle is agrochemically acceptable, appropriate for application to plants or in the vicinity of plants, without damaging them.
The present invention also relates to a nucleic acid fragment, in particular DNA, natural or synthetic, encoding the heliomicine defined above, including the “peptide-heliomicine” fusion peptide defined above. It may be according to the invention a fragment which is synthesized or which is isolated from the lepidepteron Heliothis, or alternatively a derived fragment, suitable for the expression of heliomicine in the host organism where the peptide will be expressed. The nucleic acid fragment may be obtained according to standard isolation and purification methods, or alternatively by synthesis according to the customary methods of successive hybridizations of synthetic oligonucleotides. These techniques are in particular described by Ausubel et al.
According to the present invention, “nucleic acid fragment” is understood to mean a nucleotide sequence which may be of the DNA or RNA type, preferably of the DNA type, in particular double-stranded.
According to one embodiment of the invention, the nucleic acid fragment encoding heliomicine comprises the DNA sequence described by bases 16 to 147 of the sequence identifier No. 1 (SEQ ID NO:1), or by the sequence identifier No. 2 (SEQ ID NO:2), in particular the coding portion of this sequence corresponding to bases 1 to 132, a homologous sequence or a sequence complementary to the said sequence.
According to another embodiment of the invention, the nucleic acid fragment encoding the “peptide-heliomicine” fusion peptide comprises the DNA sequence described by the sequence identifier No. 1 (SEQ ID NO:1) or that described by the sequence identifier No. 3 (SEQ ID NO:3), in particular the coding portion corresponding to bases 3 to 224, or that described by the sequence identifier No. 18 (SEQ ID NO:18), in particular the coding portion corresponding to bases 7 to 205, a homologous sequence or a sequence complementary to the said sequences.
“Homologue” is understood to mean according to the invention a nucleic acid fragment exhibiting one or more sequence modifications relative to the nucleotide sequence described by the sequence identifiers Nos. 1, 2 or 3 (SEQ ID NOS:1, 2 or 3) and encoding heliomicine or the “peptide-heliomicine” fusion peptide. These modifications may be obtained according to the customary mutation techniques, or alternatively by choosing the synthetic oligonucleotides used in the preparation of the said sequence by hybridization. In the light of the multiple combinations of nucleic acids which may lead to the expression of the same amino acid, the differences between the reference sequence described by the sequence identifiers Nos. 1, 2 or 3 (SEQ ID NOS:1, 2 or 3) and the corresponding homologue may be substantial, all the more so since small-sized DNA fragments arc involved which can be produced by chemical synthesis. Advantageously, the degree of homology will be at least 70% compared with the reference sequence, preferably at least 80%, more preferably at least 90%. These modifications. are generally neutral, that is to say that they do not affect the primary sequence of the resulting heliomicine or fusion peptide.
The present invention also relates to a chimeric gene (or expression cassette) comprising a coding sequence as well as heterologous regulatory elements at the 5′ and 3′ positions capable of functioning in a host organism, in particular plant cells or plants, the coding sequence comprising at least one DNA fragment encoding heliomicine or the “peptide-heliomicine” fusion peptide as defined above.
Host organism is understood to mean any lower or higher, mono- or pluricellular organism into which the chimeric gene according to the invention may be introduced, for the production of heliomicine. It includes in particular bacteria, for example E. coli, yeasts, in particular of the genera Saccharomyces or Kluyveromyces, Pichia, fungi, in particular Aspergillus, a baculovirus, or preferably plant cells and plants.
“Plant cell” is understood to mean according to the invention any cell derived from a plant and capable of constituting undifferentiated tissues such as calli, differentiated tissues such as embryos, plant portions, plants or seeds.
“Plant” is understood to mean according to the invention any differentiated multicellular organism capable of photosynthesis, in particular monocotyledonous or dicotyledonous plants, more particularly crop plants intended or otherwise as animal or human food, such as maize, wheat, rape, soyabean, rice, sugarcane, beet, tobacco, cotton and the like.
The regulatory elements necessary for the expression of the DNA fragment encoding heliomicine are well known to persons skilled in the art according to the host organism. They comprise in particular promoter sequences, transcription activators, terminator sequences, including start and stop codons. The means and methods for identifying and selecting the regulatory elements are well known to persons skilled in the art.
For the transformation of microorganisms such as yeasts or bacteria, the regulatory elements are well known to persons skilled in the art, and comprise in particular promoter sequences, transcription activators, transit peptides, terminator sequences and start and stop codons.
To direct the expression and the secretion of the peptide in the yeast culture medium, a DNA fragment encoding heliomicine is integrated into a shuttle vector which comprises the following elements:    markers which make it possible to select the transformants. Preferably, the ura-3 gene is used for yeast and the gene which confers resistance to ampicilline for E. coli,     a nucleic sequence allowing the replication (replication origin) of the plasmid in yeast. Preferably, the replication origin of the yeast 2i plasmid is used,    a nucleic sequence allowing the replication (replication origin) of the plasmid in E. coli,     an expression cassette consisting
(1) of a promoter regulatory sequence. Any promoter sequence of a gene which is naturally expressed in yeast may be used. Preferably, the promoter of the S. cerevisiae Mfα1 gene is used.
(2) of a sequence encoding a signal peptide (or prepeptide) in combination with a homing peptide (or propeptide). These regions are important for the correct secretion of the peptide. Preferably, the sequence encoding the pre-pro-peptide of the precursor of factor Mfα1 is used.
(3) of a polyadenylation or terminator regulatory sequence. Preferably, the terminator of S. cerevisiae phosphoglycerate kinase (PGK) is used. In the expression cassette, the sequence encoding heliomicine is inserted downstream of the pre-pro-sequence and upstream of the PGK terminator.
These elements have been described in several publications including Reichhart et al., 1992, Invert. Reprod. Dev., 21, pp 15-24 and Michaut et al., 1996, FEBS Letters, 395, pp 6-10.
Preferably, yeasts of the S. cerevisiae species are transformed with the expression plasmid by the lithium acetate method (Ito et al., 1993, J. Bacteriol, 153, pp 163-168). The transformed yeasts are selected on a selective agar medium which does not contain uracil. The mass production of transformed yeasts is carried out by culturing for 24 h to 48 h in a selective liquid medium.
The transformation of microorganisms makes it possible to produce heliomicine on a larger scale. The present invention therefore also relates to a method of preparing heliomicine, comprising the steps of culturing a transformed microorganism comprising a gene encoding heliomicine as defined above in an appropriate culture medium, followed by the extraction and total or partial purification of the heliomicine obtained.
Preferably, during the extraction of the heliomicine produced by yeasts, the yeasts are removed by centrifugation and the culture supernatant is placed in contact with an acidic solution which may be a solution of an inorganic or organic acid, such as for example hydrochloric acid or acetic acid. The extract obtained is then centrifuged at cold temperature at a speed of 4000 to 10,000 rpm at 4° C. for 30 to 60 min.
The purification of heliomicine may be preceded by a step of fractionation of the supernatant obtained following the extraction step. Preferably, during the fractionation step, the extract is deposited on the reversed phase in order to carry out a solid phase extraction. The washing of the molecules which arc soluble in water is carried out with a dilute acidic solution and the elution of the hydrophobic molecules with an appropriate eluant. Good results are obtained with trifluoroacetic acid for the washing and an eluant containing increasing quantities of acetonitrile in dilute acidic solution.
Preferably, the purification of heliomicine is carried out in two stages: a cation-exchange hihg-performance liquid chromatography (HPLC) followed by a reversed phase HPLC with a suitable eluant which may be different from or identical to that of the preceding phase. The various steps of the purification are monitored by a test of inhibition of fungal growth in liquid medium. Preferably, the test is carried out with the fungus Neurospora crassa. 
The sequence of the heliomicine produced by the transformed yeasts is analyzed according to the method of sequencing by Edman degradation and by mass spectrometry. The structural characterization is carried out directly on the peptide produced, on the peptide modified by reduction/alkylation as well as on fragments of the peptide. The peptide sequence and the molecular mass of the heliomicine produced were compared with those of the native heliomicine extracted from the haemolymph of H. virescens. The results show that the two molecules have the same primary structure. The determination of the position of the disulphide bridges indicates that the arrangement of the disulphide bridges is identical in both peptides, the native peptide and the one produced by the transformed microorganism.
The invention relates more particularly to the transformation of plants. As promoter regulatory sequence in plants, it is possible to use any promoter sequence of a gene which is naturally expressed in plants, in particular a promoter of bacterial, viral or plant origin such as, for example, that of a gene for the small subunit of ribulose-biscarboxylase/oxygenase (RuBisCO) or of a plant virus gene such as, for example, that of the cauliflower mosaic (19S or 35S CAMV), or a promoter which is inducible by pathogens such as the tobacco PR-1α, it being possible to use any known suitable promoter. Preferably, a promoter regulatory sequence is used which promotes the overexpression of the coding sequence constitutively or induced by attack by a pathogen, such as for example that comprising at least one histone promoter as described in application EP 0,507,698.
According to the invention, it is also possible to use, in combination with the promoter regulatory sequence, other regulatory sequences which are situated between the promoter and the coding sequence, such as transcription activators (enhancer), such as for example the translation activator of the tobacco mosaic virus (MTV) which is described in application WO 87/07644, or of the tobacco etch virus (TEE) which is described by Carrington & Freed.
As polyadenylation or terminator regulatory sequence, there may be used any corresponding sequence of bacterial origin, such as for example the Agrobacterium tumefaciens nos terminator, or alternatively of plant origin, such as for example a histone terminator as described in application EP 0,633,317.
According to the present invention, the chimeric gene may also be combined with a selectable marker suitable for the transformed host organism. Such selectable markers are well known to persons skilled in the art. They may include a gene for resistance to antibiotics, or alternatively a gene for tolerance to herbicides for plants.
The present invention also relates to a cloning or expression vector for the transformation of a host organism containing at least one chimeric gene as defined above. This vector comprises, in addition to the above chimeric gene, at least one replication origin. This vector may consist of a plasmid, a cosmid, a bacteriophage or a virus, which are transformed by the introduction of the chimeric gene according to the invention. Such transformation vectors, according to the host organism to be transformed, are well known to persons skilled in the art and are widely described in the literature.
For the transformation of plant cells or of plants, they may include in particular a virus which may be used for the transformation of developed plants and which contains in addition its own elements for replication and expression. Preferably, the vector for transforming plant cells or plants according to the invention is a plasmid.
The subject of the invention is also a method of transforming host organisms, in particular plant cells by integration of at least one nucleic acid fragment or a chimeric gene as defined above, which transformation may be obtained by any appropriate known means widely described in the specialized literature and in particular the references cited in the present application, more particularly using the vector according to the invention.
A series of methods consists in bombarding cells, protoplasts or tissues with particles to which DNA sequences are attached. Another series of methods consists in using, as means of transfer into plants, a chimeric gene inserted into an Agrobacterium tumefaciens Ti or Agrobacterium rhizogenes Ri plasmid.
Other methods may be used, such as microinjection or electroporation, or alternatively direct precipitation by means of PEG.
Persons skilled in the art will make the choice of the appropriate method according to the nature of the host organism, in particular of the plant cell or of the plant.
The subject of the present invention is also the host organisms, in particular plant cells or plants, transformed and containing an effective quantity of a chimeric gene comprising a coding sequence for heliomicine defined above.
The subject of the present invention is also the plants containing transformed cells, in particular the plants regenerated from the transformed cells. There generation is obtained by any appropriate means which depends on the nature of the species, as described for example in the above references.
For the methods of transforming plant cells and of regenerating plants, there may be mentioned in particular the following patents and patent applications: U.S. Pat. Nos. 4,459,355, 4,536,475, 5,464,763, 5,177,010, 5,187,073, EP 267,159, EP 604,662, EP 672,752, U.S. Pat. Nos. 4,945,050, 5,036,006, 5,100,792, 5,371,014, 5,478,744, 5,179,022, 5,565,346, 5,484,956, 5,508,468, 5,538,877, 5,554,798, 5,489,520, 5,510,318, 5,204,253, 5,405,765, EP 442,174, EP 486,233, EP 486,234, EP 539,563, EP 20674,725, WO 91/02701 and WO 95/06128.
The present invention also relates to the transformed plants derived from the cultivation and/or crossing of the above regenerated plants, as well as the seeds of transformed plants.
The plants thus transformed are resistant to certain diseases, in particular to certain fungal or bacterial diseases. As a result, the DNA sequence encoding heliomicine may be integrated with the main objective of producing plants resistant to the said diseases, heliomicine being effective against fungal diseases such as those caused by Cercospora, in particular Cercospora beticola, Cladosporium, in particular Cladosporium herbarum, Fusarium, in particular Fusarium culmorum or Fusarium graminearum, or by Phytophthora, in particular Phytophihora cinnamomi. 
The chimeric gene may also comprise, and advantageously, at least one selectable marker, such as one or more genes for tolerance to herbicides.
The DNA sequence encoding heliomicine may also be integrated as a selectable marker during the transformation of plants with other sequences encoding other peptides or proteins of interest, such as for example genes for tolerance to herbicides.
Such genes for tolerance to herbicides are well known to persons skilled in the art and are in particular described in patent applications EP 115,673, WO 87/04181, EP 337,899, WO 96/38567 or WO 97/04103.
Of course the transformed cells and plants according to the invention may comprise, in addition to the sequence encoding heliomicine, other heterologous sequences encoding proteins of interest such as other additional peptides which are capable of conferring on the plant resistance to other diseases of bacterial or fungal origin, and/or other sequences encoding proteins for tolerance to herbicides and/or other sequences encoding proteins for resistance to insects, such as the Bt proteins in particular.
The other sequences may be integrated by means of the same vector comprising a chimeric gene, which comprises a first sequence encoding heliomicine and at least one other sequence encoding another peptide or protein of interest.
They may also be integrated by means of another vector comprising at least the said other sequence, according to the customary techniques defined above.
The plants according to the invention may also be obtained by crossing parents, one carrying the gene according to the invention encoding heliomicine, the other carrying a gene encoding at least one other peptide or protein of interest.
Among the sequences encoding other antifungal peptides, there may be mentioned that encoding drosomycin, which is described in patent application FR 2,725,992 and by Fehlbaum et al.(1994), and in unpublished patent application FR 97 09115 filed on 24 Jul. 1997, or that encoding androctonin which is described in patent application FR 2,745,004 and in unpublished patent application FR 97 10362 filed on 20 Aug. 1997.
The present invention finally relates to a method of cultivating transformed plants according to the invention, the method consisting in planting the seeds of the said transformed plants in a plot of a field appropriate for cultivating the said plants, in applying to the said plot of the said field an agrochemical composition, without substantially affecting the said seeds or the said transformed plants, then in harvesting the cultivated plants when they arrive at the desired maturity and optionally in separating the seeds from the harvested plants.
Agrochemical composition is understood to mean according to the invention any agrochemical composition comprising at least one active product having one of the following activities: herbicide, fungicide, bactericide, virucide or insecticide.
According to a preferred embodiment of the method of cultivation according to the invention, the agrochemical composition comprises at least one active product having at least one fungicidal and/or bactericidal activity, more preferably exhibiting an activity which is complementary to that of the heliomicine produced by the transformed plants according to the invention.
Product exhibiting an activity which is complementary to that of heliomicine is understood to mean according to the invention a product exhibiting a complementary activity spectrum, that is to say a product which will be active against attacks by contaminants (fungi, bacteria or viruses) which are not sensitive to heliomicine, or alternatively a product whose activity spectrum covers that of heliomicine, completely or in part, and whose dose for application will be substantially reduced because of the presence of the heliomicine produced by the transformed plant.
The examples below make it possible to illustrate the present invention without however limiting its scope.